Apparatus for preparing cDNA libraries from single cells

Patent metadata

Field	Value
Publication number	US-10328428-B2
Application number	US-201715406451-A
Country	US
Kind code	B2
Filing date	Jan 13, 2017
Priority date	Oct 2, 2002
Publication date	Jun 25, 2019
Grant date	Jun 25, 2019

Abstract

Nucleic acid from cells and viruses sampled from a variety of environments may purified and expressed utilizing microfluidic techniques. In accordance with one embodiment of the present invention, individual or small groups of cells or viruses may be isolated in microfluidic chambers by dilution, sorting, and/or segmentation. The isolated cells or viruses may be lysed directly in the microfluidic chamber, and the resulting nucleic acid purified by exposure to affinity beads. Subsequent elution of the purified nucleic acid may be followed by ligation and cell transformation, all within the same microfluidic chip. In one specific application, cell isolation, lysis, and nucleic acid purification may be performed utilizing a highly parallelized microfluidic architecture to construct gDNA and cDNA libraries.

First claim

What is claimed is:

1. A microfluidic apparatus comprising:
(a) a solid substrate;
(b) a cell separation and processing system embedded in the substrate that includes an arrangement of flow channels configured so that individual cells from a cell population can be singly isolated;
(c) one or more inlet channels in the substrate connected with the cell separation and processing system so that a reagent can be delivered through the inlet channel(s) and combined with cells isolated by the system; and
(d) one or more outlet channels in the substrate connected with the cell separation and processing system so that contents obtained by lysing isolated cells can be flowed through the outlet channel(s) and kept separate;
wherein the apparatus is designed and constructed so that a user can prepare a plurality of cDNA libraries from single cells by a method that comprises:
(1) receiving a sample of cells into the cell separation and processing system of the apparatus;
(2) processing the sample in the cell separation and processing system such that a plurality of single cells from the sample are fluidically isolated from all other cells in the sample, thereby producing isolated single cells;
(3) combining a lysing chemical or buffer with the isolated cells such that mRNA is liberated from each of at least some of the isolated single cells in a manner that keeps the mRNA from each of the isolated single cells separate; and
(4) reverse transcribing and amplifying the mRNA liberated in step (3) such that a separate cDNA library is formed from at least some of the isolated single cells;
thereby producing said plurality of cDNA libraries from single cells.

2. The microfluidic apparatus of claim 1 , wherein the method further comprises:
(5) flowing the separate cDNA libraries through the outlet channel(s) of the apparatus and keeping separate at least some of the cDNA libraries formed in step (4).

3. The microfluidic apparatus of claim 1 , wherein step (2) and step (3) are performed separately.

4. The microfluidic apparatus of claim 1 , wherein the cell separation and processing system is configured to singly isolate cells from the cell population by way of a plurality of valves in the system that are operable to close channels between single cells.

5. The microfluidic apparatus of claim 1 , wherein the cell separation and processing system is configured to singly isolate bacteria.

6. The microfluidic apparatus of claim 1 , wherein the cell separation and processing system is configured to singly isolate eukaryotic cells.

7. The microfluidic apparatus of claim 1 , further comprising one or more mixing structures configured to actively mix a lysing agent delivered through the inlet channels with cells singly isolated by the cell separation and processing system.

8. The microfluidic apparatus of claim 7 , wherein the one or more mixing structures are rotary mixers.

9. The microfluidic apparatus of claim 1 , wherein step (3) comprises operating the apparatus so as to actively mix the lysis chemical or buffer with the single cells.

10. The microfluidic apparatus of claim 1 , wherein step (3) comprises operating the apparatus so as to diffusively mix the lysis chemical or buffer with the single cells.

11. The microfluidic apparatus of claim 1 , configured for sequence analysis of cDNA libraries from the single cells.

12. A microfluidic apparatus comprising:
(a) a solid substrate;
(b) a cell separation and processing system embedded in the substrate that includes an arrangement of flow channels configured so that individual cells from a cell population can be singly isolated;
(c) one or more inlet channels in the substrate connected with the cell separation and processing system so that a reagent can be delivered through the inlet channel(s) and combined with cells isolated by the system; and
(d) one or more outlet channels in the substrate connected with the cell separation and processing system so that contents obtained by lysing isolated cells can be flowed through the outlet channel(s) and kept separate;
wherein the apparatus is designed and constructed so that a user can prepare a plurality of nucleic acid libraries from single cells by a method that comprises:
(1) receiving a sample of cells into the cell separation and processing system of the apparatus;
(2) processing the sample in the cell separation and processing system such that a plurality of single cells from the sample are fluidically isolated from all other cells in the sample, thereby producing isolated single cells;
(3) combining a lysing chemical or buffer with the isolated cells such that nucleic acid is liberated from each of at least some of the isolated single cells in a manner that keeps the nucleic acid from each of the isolated single cells separate; and
(4) amplifying the nucleic acid liberated in step (3) such that a separate nucleic acid library is formed from at least some of the isolated single cells;
thereby producing said plurality of nucleic acid libraries from single cells.

13. The microfluidic apparatus of claim 12 , wherein the method further comprises:
flowing the separate nucleic acid libraries through the outlet channel(s) of the apparatus and keeping separate at least some of the nucleic acid libraries formed in step (4).

14. The microfluidic apparatus of claim 12 , wherein step (2) and step (3) are performed separately.

15. The microfluidic apparatus of claim 12 , wherein the cell separation and processing system is configured to singly isolate cells from the cell population by way of a plurality of valves in the system that are operable to close channels between single cells.

16. The microfluidic apparatus of claim 12 , further comprising one or more mixing structures configured to actively mix a lysing agent delivered through the inlet channels with cells singly isolated by the cell separation and processing system.

17. The microfluidic apparatus of claim 16 , wherein the one or more mixing structures are rotary mixers.

18. The microfluidic apparatus of claim 12 , wherein step (3) comprises operating the apparatus so as to actively mix the lysis chemical or buffer with the single cells.

19. The microfluidic apparatus of claim 12 , wherein step (3) comprises operating the apparatus so as to diffusively mix the lysis chemical or buffer with the single cells.

20. The microfluidic apparatus of claim 12 , configured for sequence analysis of nucleic acid or cDNA libraries from the single cells.

Assignees

• California Inst Of Techn

Inventors

• Hong Jong Wook
• Studer Vincent
• Anderson W French
• Quake Stephen R
• Leadbetter Jared

Classifications

• B01L2300/1827

  using resistive heater · CPC title

• B01L3/50273 Primary

  characterised by the means or forces applied to move the fluids · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• B01L2400/0481

  squeezing of channels or chambers · CPC title

• B01L2400/0655

  pinch valves · CPC title